siRNA-mediated knock-down of endogenous Hrs affects Ag-induced FcεRI trafficking.

RBL-2H3 cells were transfected with Ctrl- or Hrs-siRNA. (A) After 24 h total cell lysates were analyzed by Western blotting with the indicated Abs. (B and C) Cells were sensitized and stimulated as in Figure 5C for 1 h (B) or 2 h (C), incubated with a FITC-conjugated anti-IgE to visualize IgE-FcεRI complexes (green), stained (red) for EEA1 to identify early endosomes (B) or with Lyso-Tracker (C) to identify late endosomes and lysosomes, and analized with an ApoTome microscope. Red and green images were collected and merged, with yellow coloration indicating colocalization. The differential interference contrast (DIC) image overlay the fluorescence image is also shown. Scale bar indicates 10 µm. Results are representative of three independent experiments. (D) Quantitative analysis of the results shown in B and C. The mean percent of colocalization was calculated analyzing at least 40 cells randomly taken at each time-point from each experiments. Asterisks indicate statistical significance of p<0.001 with respect to control.